Image cytometry has been increasingly used in cytopathology and histopathology to detect aneuploid cell populations within preneoplastic or neoplastic lesions. DNA image cytometry has gained wide acceptance in pathology and cytopathology as a means to obtain diagnostic and prognostic information for human cancer. Such diagnoses require the accurate determination of cellular DNA content. Accordingly, various methods for quantitatively staining nuclear DNA have been developed. The reproducibility and reliability of several quantitative DNA stains has been recently reported. "A Comparative Study of Quantitative Stains for DNA in Image Cytometry," Mickel, U. V. and Becker, Jr., R. L., Analytical and Quantitative Cytology and Histology 1991, 13:253-260.
Included among the various techniques for staining cellular DNA are Feulgen staining techniques. Feulgen, R. and Voit K., Z. Physiol. Chem. 1924, 136:57-61. Feulgen discovered that hydrolysis of fixed tissues (i.e., DNA hydrolysis) exposed the deoxypentose present in cell nuclei in an aldehyde form. Subsequently, Feulgen found that the mild acid hydrolysis of cellular DNA followed by the addition of a Schiff reagent provided a reddish-purple color to DNA-containing structures. The Feulgen technique continues to be practiced as a method for quantitating cellular DNA and generally involves the steps of oxidizing nucleic acids to provide aldehydes, and reacting these aldehydes with a Schiff reagent to form a purple-red color indicative of the presence of DNA. Because the reaction is stoichiometric, the intensity of the color is directly related to the amount of DNA in the sample, provided that excess reagents are washed out.
In 1954, a thionin-sulfite reagent was found to exclusively stain nuclei of hydrolyzed sections of mouse kidney and liver cell nuclei. Van Duijn, P., J. Histochem. Cytochem. 1956, 4:55-63. The aldehyde reagent contained thionin and sulfur dioxide in a medium of t-butanol and water. This thionin reagent was prepared by acidifying a solution of thionin in aqueous t-butanol (water:t-butanol, 1:1) with aqueous hydrochloric acid followed by the addition of sodium metabisulfite. Van Duijn concluded from the histochemical data that the thionin-sulfite/t-butanol reagent contains one or more components that react with aldehydes, although the exact chemical nature of the reaction between thionin and sulfur dioxide and the nucleic acids was unclear. The use of Feulgen staining methods has continued to present, and various compounds have been used as Feulgen stains. Thionin, generally regarded as a nuclear stain, is one of the more commonly used stains in the Feulgen procedure.
Despite the many years that have passed since the original report of the thionin-Feulgen stain reagent, the composition of the reagent has remained unchanged (i.e., a solution of thionin and sodium metabisulfite in aqueous t-butanol adjusted to about pH 1.5 with aqueous hydrochloric acid).
Although the Feulgen thionin staining method developed in the 1950s does facilitate quantitative DNA measurements, the use of t-butanol as a solvent for the traditional thionin staining reagent is not without its drawbacks. First, t-butanol is an irritating substance and presents a work hazard. Second, because t-butanol has a melting point of about 25.degree. C., it is often a solid at room temperature and requires heating and melting so that it may be dispensed as a liquid in the preparation of the reagent. Unlike most other low molecular weight alcohols, t-butanol is difficult to dispose of properly. Because of its hazardous nature, there are also shipping restrictions associated with t-butanol. In contrast to other simple alcohols, t-butanol is extremely expensive (e.g., $45 per liter compared to methanol or ethanol at about $2 per liter). Perhaps most importantly, the useful shelf life of thionin/t-butanol staining solutions is about two days. Such a short shelf life precludes the storage and therefore commercial utility of such a reagent, and requires one who wishes to use the thionin reagent in a Feulgen staining method to prepare the reagent immediately prior to use.
Accordingly, there exists a need for a thionin-based reagent that offers the advantages of cellular DNA quantitation afforded by the traditional thionin/t-butanol staining reagent without the accompanying disadvantages associated with t-butanol, a key component of the reagent. More specifically, there exists a need for a thionin staining reagent that has a long and stable shelf life. The present invention seeks to fulfill these needs and provides further related advantages.